Delayed and sustained drug release

ABSTRACT

The invention relates to the controlled release of preparations of therapeutic agents, for example a steroid; formulations comprising said preparations; and the use of said formulations to treat diseases such as those diseases which would benefit from steroid treatment.

The invention relates to the controlled release of preparations oftherapeutic agents, for example steroids; formulations comprising saidpreparations; and the use of said formulations to treat diseases whichwould benefit from steroid treatment.

It has long been desirable to provide drugs which can be released in acontrolled manner. Controlled drug release may be viewed in two ways.Firstly, to provide a sustained drug release over a period of time sothat a whole body is not flooded with the drug when administered. Thedrug is then cleared by the body resulting in a rapid fall in systemiclevels thereby not providing adequate therapeutic effect over an entiretreatment regime. A second view of controlled drug release is thesituation when the delivery of the drug is desired at a specific timeand in a precise manner. In this case maintaining a constant systemiclevel may not be desirable and indeed result is adverse side effects.Problems therefore arise when a condition requires both sustained drugrelease but which has to be regulated in a specific manner, for examplein accordance with a circadian rhythm or menstrual cycle.

An example of a class of diseases which has both the above elements areconditions resulting from adrenal failure which result in hormonalinsufficiency and diseases which may be worse at certain times of theday such as rheumatoid arthritis and asthma.

Adrenal failure occurs in approximately 1/10,000 of the population. Itmay be due to either primary adrenal failure (e.g. Addison's diseasecommonly occurring following autoimmune damage to the adrenal gland orTB), or secondary adrenal failure (which occurs due to pituitary failurewhich may be caused by a pituitary tumour or surgery). In causes ofprimary adrenal failure ACTH levels from the pituitary will be high andin secondary adrenal failure ACTH levels are inappropriately low.Another common cause of adrenal failure is suppression of the normalpituitary-adrenal axis by steroid therapy such as that used forchemotherapy, rheumatoid arthritis and asthma. Thus, adrenal failure isa relatively common condition and many patients have to take long-termsteroid replacement therapy.

Hydrocortisone is the preferred steroid treatment for patients withadrenal failure. However, other glucocorticoids have been used includingcortisone acetate which requires conversion to cortisol in the liver,prednisolone, prednisone, and dexamethasone. Hydrocortisone is the mostcommonly used drug as it is equivalent to cortisol, is rapidly absorbedand is inexpensive. Cortisol is released from the adrenal gland underthe regulation of ACTH derived from the pituitary gland (FIG. 1). Thereis a circadian rhythm to cortisol release with high levels first thingin the morning and very low levels around midnight (FIG. 2). ACTH andthus cortisol levels begin to rise around 3 am and peak at 7 amgradually falling over the day to a nadir at midnight (Krieger et al.,1971; Ross et al., 1991). Cortisol is a steroid hormone essential forsurvival especially during stress such as infection. Deficiency incortisol results in fatigue, wasting, diarrhoea and finally deathusually with an Addisonian crisis precipitated by infection.

In treating patients with adrenal failure, an attempt is made to mimicthe cortisol circadian rhythm by giving a high dose of hydrocortisonewhen the patient wakes in the morning and then a second dose later inthe day. This treatment regimen is effective but does not reflect normalphysiology in that patients will wake with undetectable cortisol levelsand only get a peak an hour after taking their hydrocortisone (FIG. 3).

The means to regulate controlled drug release are known in the art.

For example, U.S. Pat. No. 4,261,969, which is incorporated byreference, discloses a polymer composition which is enzyme activated.The composition comprises a sensing means which can detect small amountsof a compound in a complex mixture, for example blood, which is anindicator of the body's need for the drug, and a delivery means whichsenses the change in the sensing means thereby releasing the drug at arequired time in a dose dependent manner. The system is suitable for usein the delivery of a contraceptive drug.

A further example is disclosed in EP01077065, which is incorporated byreference. The controlled release formulation comprises a drug corewhich is surrounded by a release control layer which breaksdown after apredetermined delay. EP01077065 also discloses a drug release layeroutside the release control layer which provides for an initial rapidrelease followed by the release of drugs from the drug core. Thisprovides for the delivery of at least two drug doses in a delayedmanner.

U.S. Pat. No. 6,207,197, which is incorporated by reference, discloses apharmaceutical composition which is adapted to be retained in thestomach to treat diseases, such as ulcers. These are referred to asgastro-retentive drugs. The invention describes microspheres comprisingan inner core containing a therapeutic agent surrounded by a waterinsoluble polymer which is provided with an outer layer of bioadhesivecationic polymer. The adhesive polymer functions to retain themicrosphere in the stomach thereby facilitating the concentrated releaseof the therapeutic agent in the stomach.

EP01053752, which is incorporated by reference, discloses a furtherexample of a preparation which shows controlled release. The preparationcomprises two parts, a female and male part, the female part is madefrom a water insoluble polymer and the male part formed from acomposition consisting of ethyl acrylate: methyl methacrylate:trimethylammonioethyl methacrylate co-polymer and a methylacrylic:ethylacrylate co-polymer. The therapeutic agent is contained within the maleand female parts. The formulation is pH sensitive only releasing atherapeutic agent at neutral pH thereby passing through the stomachintact and only releasing in the neutral environment of the smallintestine.

WO010957, which is incorporated by reference, discloses an implant whichis provided with a coating comprising a polymer matrix which is formedfrom ethylenically unsaturated monomers which includes a zwitteronicmonomer, for example2-methacryloyloxyethyl-2′-trmethylammoniumethlyphosphate salt. Thecomposition absorbs a therapeutically active substance which is thendried by evaporation of the solvent included with the active substance.The implant is then ready for implantation into the patient and beginsto slowly release the active substance.

U.S. Pat. No. 6,217,911, which is incorporated by reference, discloses acontrolled release microcapsule for the controlled release ofnon-steroidal anti-inflammatory drugs for 24 hours to 2 months. Themicrocapsule is biocompatible and biodegradable and manufactured fromDL-lactide-co-glycolide. The composition is topically applied to softtissues surrounding a surgical incision or wound site. Typically themicrospheres are loaded with lidocaine to provide slow release painrelief.

A yet further example of a delayed release formulation is disclosed inWO02/30398, which is incorporated by reference in its entirety. Theformulation comprises a core which includes a drug and a disruptionagent and further comprises a regulatory membrane coating on the coreformed from a mixture of a water soluble gel-forming polymer and awater-insoluble film-forming polymer. The disruption agent is forexample an agent which expands on hydration (e.g.hydroxypropylcellulose, sodium starch glycolate, sodiumcarboxymethylcellulose, croscarmellose sodium or carbomer). The coreinlcudes a spheronisation aid (e.g. microcrystalline cellulose). Thewater soluble gel forming polymer of the regulatory membrane coating isa high viscosity grade hydroxyalkyllcellulose (e.g.hydroxypropylmethylcellulose) or a methyl cellulose. The water insolublefilm forming polymer of the regulatory membrane coating is an alkylcellulose (e.g. ethyl cellulose).

It is apparent that there are means to provide controlled release oftherapeutic agents. However the problem addressed by the presentinvention is how to provide a treatment regime which combines both thedelayed and sustained release of a therapeutic agent to provide anoptimal treatment regime.

The invention therefore provides a treatment regime suitable for ananimal which comprises the administration of at least one therapeuticagent and means which allow for the delayed and sustained release ofsaid therapeutic agent.

According to a first aspect of the invention there is provided a methodfor the treatment of an animal wherein a therapeutic agent isadministered which has the characteristics of controlled release.

In a preferred method of the invention said the delayed and sustainedrelease of said therapeutic agent is in accordance with the circadianrhythm of a patient who is administered said agent.

In a further preferred method of the invention an animal is administereda therapeutic agent which is released in a sustained manner which isfollowed by a therapeutic agent which is released in a delayed butsustained manner.

Controlled release is construed as, delayed release, sustained releaseor a combination of delayed and sustained release.

In a preferred method of the invention said method comprises the stepsof:

-   -   i) providing a combined preparation of a therapeutic agent and a        delivery vehicle wherein said vehicle provides for the sustained        release of the therapeutic agent;    -   ii) administering the combined preparation in (i) to an animal        requiring treatment such that the therapeutic agent is released        in a sustained manner;    -   iii) providing a combined preparation of a therapeutic agent and        a delivery vehicle wherein said vehicle provides for the delayed        but sustained release of the therapeutic agent; and    -   iv) administering the combined preparation in (iii) to an animal        requiring treatment such that the therapeutic agent is released        in a delayed but sustained manner.

In a preferred method of the invention said animal is human.

In a preferred method of the invention said therapeutic agent is asteroid.

In a further preferred method of the invention said therapeutic agent iscortisol, hydrocortisone, a glucocorticoid or functional derivativesthereof.

A patient would take a sustained release preparation in the morning anda night-time preparation which would be a delayed and sustained releaseformulation (FIG. 4). Based on pharmacokinetic modelling this twicedaily administration would reproduce the normal circadian rhythm of, forexample, cortisol production (FIG. 4).

In a preferred method according to the invention said sustained releasepreparation is 10-100 times slower than the preparation without thedelivery vehicle. Preferably said sustained release is 30-80 timesslower and more preferably still about 45-50 times slower than thepreparation without the delivery vehicle.

In a further preferred method of the invention said sustained releasepreparation is administered in the morning, preferably between 08:00 and12:00.

In a yet further preferred method of the invention said delayed andsustained release formulation is administered in the evening, preferablybetween 20:00 and 24:00.

According to a further aspect of the invention there is provided apharmaceutical composition comprising a therapeutic agent and a deliveryvehicle characterised in that the delivery vehicle provides for thesustained release of the therapeutic agent.

According to a further aspect of the invention there is provided apharmaceutical composition comprising, a therapeutic agent and adelivery vehicle characterised in that the delivery vehicle provides forthe delayed and sustained release of the therapeutic agent.

In a preferred embodiment of the invention said therapeutic agent iscortisol/hydrocortisone, a glucocorticoid or a functional derivativethereof.

When administered, the pharmaceutical compositions of the presentinvention are administered in pharmaceutically acceptable preparations.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents, such aschemotherapeutic agents.

The therapeutic agent of the invention can be administered by anyconventional route, including injection. The administration may, forexample, be oral, intravenous, intraperitoneal, intramuscular,intracavity, subcutaneous, or transdermal.

The pharmaceutical compositions of the invention are administered ineffective amounts. An “effective amount” is that amount of a compositionthat alone, or together with further doses, produces the desiredresponse. This may involve only slowing the progression of the diseasetemporarily, although more preferably, it involves halting theprogression of the disease permanently. This can be monitored by routinemethods or can be monitored according to diagnostic methods.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of cortisol/hydrocortisone,glucocorticoids or derivatives thereof for producing the desiredresponse in a unit of weight or volume suitable for administration to apatient. The response can, for example, be monitored by measuring thephysiological effects of the composition, such as a decrease of diseasesymptoms and/or measurement of ACTH levels where appropriate. Assayswill be known to one of ordinary skill in the art and can be employedfor measuring the level of the response.

The doses of the composition administered to a subject can be chosen inaccordance with different parameters, in particular in accordance withthe mode of administration used and the state of the subject (ie age,sex, weight, body mass index (BMI). Other factors include the desiredperiod of treatment. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits.

In general, doses of the composition are formulated and administered indoses between 1 mg and 30 mg, and preferably between 10 mg and 25 mg,according to any standard procedure in the art. More preferably stillsaid sustained release composition is administered between 1 mg and 30mg at night and between 1 and 15 mg in the morning

An animal as used herein, is a mammal, preferably a human, and includinga non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

When administered, the pharmaceutical compositions of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. Such preparations mayroutinely contain salts, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The pharmaceutical compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation ofcortisol/hydrocortisone or functional derivative thereof, which ispreferably isotonic with the blood of the recipient. This preparationmay be formulated according to known methods using suitable dispersingor wetting agents and suspending agents. The sterile injectablepreparation also may be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Among the acceptable solvents that may beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

The use of a combined therapeutic agent and a delivery vehicle, whereinthe delivery vehicle provides for a delayed and sustained release of asteroid for the manufacture of a medicament for use in the treatment ofa disease or condition which would benefit from the administration of asteroid.

In a preferred embodiment of the invention said disease or condition isselected from the group consisting of: adrenal dysfunction; rheumatoidarthritis; inflammatory disorders; asthma; nephritis; collagen vasculardisorders; connective tissue diseases.

Preferably the adrenal dysfunction is caused by a condition selectedfrom the group consisting of: primary or secondary adrenal failure,congenital adrenal hyperplasia, late-onset congenital adrenalhyperplasia, polycystic ovarian failure.

In a preferred embodiment of the invention adrenal dysfunction is causedby congenital adrenal dysfunction.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive conditioncommonly due to mutations in the cytochrome P450 21-hydroxylase gene(CYP21) important in the cortisol biosynthetic pathway. Thus, patientshave a deficiency in cortisol production, which leads to excess ACTHsecretion by the anterior pituitary in an attempt to increase cortisolproduction (i.e. the loss of cortisol negative feedback at thepituitary, FIG. 1). The rise in ACTH stimulates the adrenal steroidpathway but because there is a block at 21-hydroxylation there is abuild up in steroid precursors which are androgenic. This build up inandrogenic steroid precursors has important implications for the foetus,infant, child and adult with CAH.

For a female foetus the build up in androgens results in a virilisedfoetus with ambigious genitalia. In the infant and child the androgenscause pseudo-precocious puberty with excess growth and virilisation.Untreated the child will go through a very early puberty and end up veryshort. In the adult CAH is associated with infertility, virilisation ofthe female and steroid deficiency. Treatment of CAH requires steroidreplacement therapy both as replacement and to reduce androgenicprecursors. Treatment is complex as excessive steroid replacement causescomplications in its own right by reducing growth in the child andcausing thin bones and skin in the adult.

There are various treatment regimens available which attempt to provideadequate steroid levels during the day and counter the build up of ACTHat night, but none of them provide optimal therapy. The commonestregimen used in children is twice or thrice daily hydrocortisone. Thistreatment regimen provides supraphysiological levels of hydrocortisonewithin 1-2 hours of dosing (Charmandari et al., 2001) and doesn'tprevent the early morning rise in ACTH and androgenic precursors (Scottet al., 1978; Cutler, 1996). Alternative treatment regimens involvegiving a dose of steroids last thing at night which is unphysiologicaland some patients complain affects their sleeping pattern in addition towhich there is a greater risk of giving excess steroid doses as thepatient still need steroid replacement during the day.

We propose that the optimal treatment for CAH would be a delayed andsustained release hydrocortisone that could be given last thing at nightand reproduce the normal physiological pattern of cortisol secretion.

An embodiment of the invention will now be described by example only andwith reference to the following tables, figures and examples:

-   -   Table 1 illustrates variability (C.V.) and bias (% difference        from healthy matched controls of AUC) for patients on different        hydrocortisone regimes;    -   Table 2 illustrates stepwise multiple linear regression analysis        of patient variables with parameters of HC disposition;    -   Table 3 illustrates suggested hydrocortisone dosing regime;

FIG. 1 represents a schematic representation of cortisol regulation inan animal;

FIG. 2 represents the normal circadian rhythm of cortisol in an animal;

FIG. 3 represents the cortisol profile in a patient takinghydrocortisone three times during a 24 hour period; and

FIG. 4 represents a cortisol profile in a patient taking a sustained anda sustained and delayed release composition of hydrocortisone;

FIG. 5 is a graphical representation of an infusion protocol for theadministration of hydrocortisone to a patient suffering from congentialadrenal hyperplasis;

FIG. 6 is a graphical representation of ACTH and cortisol levels in apatient undergoing the infusion protocol described in FIG. 5;

FIG. 7 illustrates serum cortisol concentrations in (a) 10 fastedpatients after taking a fixed dose of 10 mg hydrocortisone, (b) 10fasted patients after taking a weight-adjusted (0.12 mg/kg) dose ofhydrocortisone, and (c) following a fixed dose of 10 mg hydrocortisone,in fasting and fed states;

FIG. 8 illustrates a range of AUCs (mean and individual data) accordingto study groups. The shaded area represents the 95% CI for observationsin the healthy control group with the mean as the continuous line;

FIG. 9 illustrates predicted AUCs based on 240 min serum cortisol versusobserved AUCs (open circles=data from patients in Study 1; closedcircles=data from patients in Study 2; bold line=trendline for Study 1patients which was subsequently used to predict AUCs of the Study 2patients; dotted line=line of identity); and

FIG. 10 illustrates (a) Circadian rhythm of serum cortisol in normalsubjects from published data (solid line) (10) and simulated cortisolprofile for a patient (broken line) following thrice dailyhydrocortisone administration according to our optimisation simulation.(b) Nomogram for individual adjustment of hydrocortisone dosage based onserum cortisol estimation 2½h-5 h after a morning HC dose taken on anempty stomach. The lines represent the 10^(th), 25^(th), 50^(th),75^(th) and 90^(th) centiles.

Materials and Methods

Hydrocortisone Infusion in a Patient with Congenital Adrenal Hyperplasia(CAH)

A treatment regime involves the following protocol. A patient begins atreatment regime at 0830 having taken normal medication the day before.An indwelling cannula is inserted into the patient and basal bloodsamples are obtained to determine basal levels of cortisol. An infusioncannula is inserted for the administration of a bolus dose ofhydrocortisone. Hydrocortisone is administered by infusion once basalsamples taken (Patient details: body weight=113 kg; iv bolus 24 mg at09:00 and follow the infusion rates changed on hourly bases). Total dosefor the 1^(st) day is 34.3 mg and 21.8 mg is given by 09:00 in the2^(nd) day). Samples are taken from the patient periodically from 0900to 0900 and sampled hourly for cortisol, and ACTH

Hydrocortisone Dosage and Administration

Dilute 100 mg of hydrocortisone in 2 ml of water for injection(concentration 1 mg/20 ul). Give bolus injection of 24 mg hydrocortisone(480 ul or 0.48 ml of hydrocortisone) using a 1 ml insulin syringe.Flush through with normal saline. For infusion put 50 mg ofhydrocortisone (1 ml) in 49 mls normal saline=1 mg/ml and the runinfusion according to the protocol above.

Assay for Cortisol in Serum Samples

Serum was separated from blood samples immediately after collection andstored at minus 20° C. until analysis. Cortisol concentrations weremeasured using an ACS:180 automatic chemiluminescence system (KironDiagnostic Corporation, East Walpole, USA). Intra- and inter-assaycoefficients of variation were less than 7.6%.

EXAMPLE 1 Cortisol Metabolism and Clearance

Cortisol secretion under basal non-stressed conditions ranges from 8-25mg/day (22-69 umol/day) with a mean of 9.2 mg/day (25 umol/day) (deLacerda et al., 1973; Gallagher et al., 1970). Cortisol althoughsecreted in the unbound state, binds to plasma proteins, cortisolbinding globulin (CBG, transcortin) and, to a lesser extent, to albumin(Hammond, 1990). Under basal conditions about 5% to 10% of circulatingcortisol is free, about 75% is bound to CBG, and the remainder is boundto albumin. However, it is the free level that is sensed and regulatedby the CRH-ACTH axis. Normal CBG has a cortisol binding capacity of 25ug/dl; increases in total plasma cortisol concentrations above thislevel result in rapid increases in levels of free cortisolconcentration. The cortisol-binding capacity of albumin is greater thanthat of CBG, but its affinity is lower. Relatively little cortisol isexcreted in the urine unchanged—less than 1%. Over 90% of cortisol andthe metabolites of cortisol are conjugated in the liver and excreted inthe urine. In normal individuals there are circadian fluctuations in thecapacity of CBG for cortisol which are lost in patients on chronicreplacement. The pharmacokinetics of 20 mg hydrocortisone have beenstudied after IV and oral administration (Derendorf et al., 1991). AfterIV administration, hydrocortisone was eliminated with a total bodyclearance of 18 L/hr and a half-life of 1.7 hr. The volume ofdistribution was 34 L. Oral bioavailablity averaged 96%. Absorption wasrapid, achieving maximum levels after 1 hour.

EXAMPLE 2 Modelling a Delayed and Sustained Release Hydrocortisone

Based on published models of circadian cortisol profiles (Chakraborty eyal. 1999) (FIG. 4) we have defined sustained and delayed releasetablets, which will provide physiological hydrocortisone therapy (FIG.4).

EXAMPLE 3 Programmable Intravenous Infusion of Hydrocortisone

Normal individuals are studied to establish that programmableintravenous infusion gives a reproducible cortisol profile. To suppressendogenous cortisol production normal individuals will be givendexamethasone 2 mg at 2200 h prior to the start of the study (Patel etal., 1984). At 2400 h a programmed infusion will be started and theindividuals will then have 20 minute sampling for 24 hours. To test thatthis regimen of hydrocortisone will suppress ACTH into the normal rangepatients with complete adrenal failure (undetectable cortisol levels)will undergo the same protocol without the administration ofdexamethasone and the measurement of ACTH.

EXAMPLE 4 Sustained/Delayed Release of Hydrocortisone

Means to provide for the sustained release of a therapeutic agentinclude, by example and not by way of limitation, changing thedissolution rate of for example, hydrocortisone, using availablemethodologies that incorporate dissolution modifying polymers into theformulation of the delivery system such that a desired rate of releaseis achieved. These may include fatty acids with different number ofcarbons, carbohydrates, and derivatives of cellulose.

Delayed release can be obtained by a variety of available methods, whichmay include the following examples. A hard impermeable capsule which issealed at the neck edge with hydrogel plug. On ingestion, the capsulebecomes exposed to gastric fluids and the water-soluble gelatin capdissolves, allowing the hydrogel plug to hydrate. At a predetermined andcontrolled time point after ingestion, the swollen plug is ejected fromthe capsule body, thereby enabling drug formulation to be released withtime of ejection controlled by the length of hydrogel plug and itsposition relative to the neck of the body.

A further example includes the use of a multilayercapsule/tablet/particulate system wherein three different polymericlayers control the time of release; an inner layer consisting ofcathionic polymer dissolving in acidic fluid, a water-solubleintermediate layer, and an outer layer consisting of enteric materialsdissolving at pH>5. After ingestion of capsule, drug release can becompletely prevented in the stomach due to the acid resistance of theouter polymeric layer. After gastric emptying, the outer layer and theintermediate layer quickly dissolve but the inner polymeric layer stillremains and effectively prevents the drug release in the intestine. Whenthe inner polymeric layer is finally dissolved by the acidic fluid thedrug content is released. The onset of the drug release, therefore, canbe controlled by the thickness of the inner polymeric layer.

A yet further example includes a so-called time control explosionsystem, consisting of 4 separate layers of seed, drug layer, swellingagent layer and water insoluble membrane. In this system, a rapid drugrelease is initiated by destruction of outer membrane. The lag time isprecisely programmed by changing the outer membrane thickness. As thedestruction of the outer membrane is caused by the water uptake of theswelling agent.

A further system comprises a swellable core material, the core beingsurrounded by an inner coat of a water-insoluble or relativelywater-insoluble coating material in which particulate water-insolublematerial is embedded. The inner coat is additionally surrounded by anouter coat that contains additional amounts of the desired agent. Whenthe delivery device enters the gastrointestinal tract, the outer coatreleases the desired agent contained therein and disintegrates, exposingthe inner coat. The particulate matter in the inner coat takes upliquid, thus forming channels interconnecting the drug-containing corewith the outside of the delivery device. Through these channels liquidenters the core which then swells to the point at which the inner coatis broken. When the integrity of the inner coat is destroyed, the corethen disintegrates, immediately releasing all or most of the drug at aspecific site. By controlling parameters in the device, such as the corematerial, carrier material in the coating, and particulate matter, thelocation of release of both pulses of the drug can be carefullycontrolled.

EXAMPLE 5 Hydrocortisone Infusion in a Patient with Congenital AdrenalHyperplasia (CAH)

The following experiment was undertaken to demonstrate that a circadianrhythm hydrocortisone infusion simulating a delayed and sustainedrelease formulation of hydrocortisone can control ACTH in an individualsuffering from CAH. The method comprised the infusion of a patientsuffering from CAH with hydrocortisone and the measurement of cortisoland ACTH levels. The patient is a man aged 34 years with CAH who iscurrently treated with oral hydrocortisone.

The infusion protocol for the patient is described in FIG. 5. FIG. 6shows that in the patient with CAH on conventional therapy the ACTH isvery high at 0900 when the cortisol is low. During the infusion the ACTHrapidly falls and the overnight increase in cortisol simulating thedelayed and sustained release hydrocortisone prevents the high morningACTH. Concomitant with the fall in ACTH the 17-OH progesterone fellfrom >500 nmol/l to a nadir of 67 nmol/l.

EXAMPLE 6 Case Studies

The study was carried out in two groups of adrenal-insufficientpatients, and a group of healthy subjects. It was approved by the NorthSheffield Local Research Ethics Committee, and all participants gavewritten informed consent. All patients had primary or secondary adrenalinsufficiency based on a 0900 h serum cortisol level off treatment ofless than 50 nmol/l. Normal controls were matched to the second group ofpatients for gender, age, height, weight and BSA within 10% ofindividual patients. Patients on oestrogen replacement were studiedafter having stopped oestrogen for 6 weeks.

Study 1—Pharmacokinetics of 10 mg fixed dose oral HC: Ten patients (5M,5F, Age 32-72 yrs, BMI 21.7-35.8 kg/m²) attended the ProgrammedInvestigation Unit on 3 occasions. They discontinued HC replacement fromnoon on the day prior to the study and fasted from midnight. On thefirst and third occasions, the patients fasted throughout the study and,on the second occasion, they received a standard breakfast 20-30 minutesbefore receiving HC. On each of the 3 study days, patients received 10mg of HC (Hydrocortone™, Merck, Sharp and Dohme) orally, between0800-0900 h. Peripheral venous blood samples were taken immediatelybefore dosage and at 20, 40, 50, 60, 70, 80, 90, 100, 120, 180, 240, 300and 360 minutes after dosage; the serum was collected and stored pendingassay for HC.

Study 2—Pharmacokinetics of individually tailored dose of oral HC andtesting of monitoring protocol: A further 10 patients (6M, 4F; 46-68 y;BMI 24.4-35.5 kg/m²) were studied after receiving HC doses of 5.5 mg/m²BSA (to the nearest practical dose unit) at 0830 h, 1 h beforebreakfast. The dosage was derived from analysis of the Study 1 data tominimise inter-subject variability. A tablet cutter was used to adjustthe HC dose in 2.5 mg quanta, i.e. quarters of 10 mg tablets. Thus, theabsolute dose ranged from 7.5 to 12.5 mg. Blood sampling was as inStudy 1. Serum concentrations of endogenous cortisol were measured in 7healthy subjects (3M, 4F; 44-68 y; BMI 23.2-28.7 kg/m²) at the same timepoints as the patients but without ingestion of HC.

Initial analysis of study 2 showed that if the dose had been adjustedfor weight (0.12 mg/kg), the variability for C_(max) and AUC could havebeen reduced further. Owing to the discrete nature of dose increments(multiples of 2.5 mg) 9 of the 10 patients who had taken theBSA-adjusted HC dose would still require the same dose if this had beenweight-adjusted. Only one of the 10 patients needed a reduction in theHC dose from 12.5 mg to 10.0 mg to be in the nearest weight-adjusteddose range.

Therefore, in the part of our analysis that was related to bodyweight-adjusted doses, the serum cortisol levels of this patient werenormalised to a 10.0 mg dose assuming concentration-doseproportionality.

Kinetic analysis of the serum cortisol concentration-time profiles inthe patients was carried out using the software package, P-Pharm(version 1.5, InnaPhase, France), assuming mono-exponential disposition,first-order absorption, and a baseline correction to account for anyendogenous levels of cortisol. Values of oral clearance (CL/F), volumeof distribution (V/F), absorption rate constant (ka) and absorption lagtime (t_(lag)) were obtained from the model fitting and were used toderive other parameter values. Bias in dosing was assessed as thepercentage difference in AUC values in patients compared to the AUC ofendogenous cortisol in the healthy subjects over the same time period.Variability in the features of the serum cortisol concentration-timeprofile in the patients was expressed as coefficient of variation (CV).The effect of food intake in Study 1 was assessed using ANOVA,accounting for repeat individual measurements in the fasting state. Bodysurface area, weight, height, BMI, dose of HC, gender and age were alsoinvestigated as co-variables. All statistical analyses were carried outusing SPSS version 10.0 (SPSS Inc, USA).

To assess the optimal dosage regimen of HC in patients with adrenalinsufficiency, simulations were carried out using the Solver tool inMicrosoft Excel™, with the aim of minimizing the residual sum of squaresbetween the simulated 24 h serum HC profile and the measured 24 hprofiles reported in the literature (Chachraborty et al 1999).Constraints on the simulated dosage regimen were a maximum of 3 dosesbetween 0600 h and 2200 h and a minimal 2.5 mg increment of HC dosage(compatible with quartering of the available 10 mg tablet).

Based on the results of Studies 1 and 2, Monte Carlo simulations werecarried out in virtual patients (n=1000; 32-72 y), using demographicinformation available for the UK population (Prescott-Clarke, 1996) toconstruct a nomogram showing the likelihood of the expected serumcortisol concentration at 2.5-5 h after early morning intake of HC.Mathcad (Ver. 6; MathSoft, Edinburgh) was used to generate randomnumbers and to run the simulations.

EXAMPLE 7 Variables Affecting Cortisol Kinetics

There was considerable variability in C_(max) and AUC values in patientsreceiving a fixed 10 mg oral dose of HC taken in the fasted state (FIG.7 a; Table 1). This variability was significantly decreased when a BSA-or weight-adjusted dose of HC was assumed (FIG. 7 b; Table 1). Weight,height and BSA were major variables affecting HC kinetics; and HC dosealso made a significant contribution (Table 2). Of these co-variates,weight had the greatest effect on HC clearance. Compared tointer-subject variability, within-subject variability was low, and thetwo mean cortisol profiles measured in the fasted state weresuperimposable (FIG. 7 c). Food intake prior to HC ingestion prolongedits absorption half-life (mean±S.E.M. fed vs fast, 43±12 vs 15±4 min;p=0.002) and decreased the oral clearance (0.226±0.031 vs 0.270±0.027L/min, p=0.05). There was an increase in the variability in C_(max) (36vs 31%,) and reduction in the variability in AUC (45 vs 50%) when HC wastaken in the fed compared to the fasted state although these changeswere not statistically significant.

HC Exposure in Patients Relative to Controls

The AUC of exogenous HC in patients compared to the endogenous AUC inhealthy control subjects indicated the least bias when dosage wasadjusted for weight (FIG. 8; Table 1).

Single Point Prediction of AUC

Correlation coefficients between the observed AUC values in Study 1patients and those predicted from their serum cortisol concentrations atspecific times were: 240 min (r=0.885, p<0.001), 40+240 min (r=0.912,p<0.001), 60+180+240 min (r=0.950, p<0.001), and 40+60+180+240 min(r=0.950, p<0.001). Thus, 78% of the variability in AUC was explained bythe serum cortisol concentration at 240 min. The predicted AUC based onthis sample was concordant with observed values in both the original set(30 samples from 10 patients in Study 1) and in the independent group of10 patients in Study 2 (FIG. 9).

Simulation of Cortisol Dosage Regimens

The serum cortisol profiles after HC administration in studies 1 & 2were compared to the mean profile of endogenous cortisol in our controlsubjects. The effects of using different fixed doses (7.5-12.5 mg) weresimulated using the observations on the 10 mg dose. Treatment with a 10mg fixed dose of HC in the fasting state was associated with a 6.3%over-exposure of patients to cortisol compared to our group of controls,and this was greater still in the fed state (24.5%). Individualisationof the dose by weight-adjustment (0.12 mg/kg) resulted in a much smallerbias (4.6%) (Table 1).

The data used for the above analysis refers to the first morning dose ofHC. The total daily dose and regimen of administration was estimatedusing 24 cortisol profiles reported in the literature (Chachraborty etal 1999) and the pharmacokinetic parameters defined by our study. Theresults show that thrice daily administration given on a dose by weightbasis provides optimal replacement within the constraints of the currentHC formulation (Table 3 and FIG. 10 a).

Nomogram to Adjust Dosage Based on Serum Cortisol Concentration

The predicted population distribution of the serum cortisolconcentration-time profile from 150 to 300 min after a 0.12 mg/kg doseof HC is shown in FIG. 10 b. This forms the basis of a nomogram toadjust individual HC dosage using a single serum sample. Thus, acortisol level falling within the 25^(th)-75^(th) centiles suggests thatthe HC dose taken by the patient results in the predicted cortisolprofile (AUC).

Therefore, the dose is appropriate for the patient. If a cortisol levelfalls above the 75^(th) centile, the HC dose may need to be reduced,whereas a cortisol level that falls below the 25^(th) centile indicatesthat the patient may require further investigation with regard topossible malabsorption, and the HC dose may need to be increased.

We find that body weight is an important variable determining HCclearance and that prescribing a fixed dose to all patients result ingreater under- and over-treatment (bias) compared to a dose adjusted bypatient's weight (bias of <5%). Weight-adjusted HC dosage (0.12 mg/kgfor the morning dose) produces serum cortisol profiles with lessinter-patient variability and is associated with AUC values similar tothe endogenous cortisol levels of matched controls during the same studyperiod (0800 h-1400 h). Characterisation of full serum cortisol profilesafter a single HC dose is done in some clinics (Feek et al 1981).However, this approach is expensive and time consuming. Based on ourdata, a single measurement of cortisol at 240 min following HC ingestioncan reliably predict the cortisol AUC.

We have modelled HC replacement regimens to provide cortisol profiles asclose to physiological levels as practical within the constraints of thecurrent HC preparation (FIG. 4; Table 3). On this basis, we recommend athrice-daily treatment regimen. The total daily dose (15 mg HC) for a 70kg patient (175 cm height) is approximately 8.1 mg/m²/day, which issimilar to the estimated cortisol production rate (Linder et al 1990).Clinicians generally give a higher replacement dose assuming incompleteabsorption and first pass hepatic metabolism (Ten et al 2001). However,the absorption of HC is rapid and oral bioavailability is almostcomplete (94-96%) (Derendorf et al 1991; Charmandri et al 2001). Ourproposed treatment regimen is based on pharmacokinetic studies performedusing an early morning dose of HC. Although HC bioavailability in theevening is likely to be the same, clearance may be lower at this time(Charmandri et al 2001). Our total daily and individual HC doses arelower than those currently used by many clinicians on the assumptionthat HC replacement therapy should be an attempt to replicate normalphysiology. It is possible that, because current preparations of HC donot allow normal physiological replacement (cortisol levels are lowfirst thing in the morning), that patients require supra-physiologicallevels of cortisol at other times of the day. We suggest that changes inHC replacement require careful monitoring including specific questioningabout fatigue.

The primary site of cortisol metabolism is the liver (Gower et al 1984).The high oral bioavailability of HC indicates that it has a low hepaticextraction ratio (i.e. the fraction of the dose escaping first-passhepatic clearance is high) and, for this reason, hepatic enzyme activityis not an important determinant of oral bioavailability. We found thatboth weight and BSA can predict HC clearance. As predictions usingweight were better, and as using weight for dose adjustment in theclinic is easier, we recommend treatment regimens based on weight. Theage range for our patients was 32-72 years, and within this range we didnot find any additional effects of age on HC pharmacokinetics apart fromthose determined by weight or BSA. It has been reported that cortisolclearance and volume of distribution increase in parallel at pubertyleaving cortisol half-life relatively unaffected (Charmandari et al,2001). Age is associated with changes in cortisol profiles in responseto stress, but mean and integrated cortisol levels are similar in youngand elderly normal subjects (Bergendahl et al 2000).

Our results demonstrate that food delays the absorption of HC andincreases the variability. This is consistent with previous reports(Barbhaiya et al 1982). In practice, patients are usually encouraged totake their first dose of HC on waking in the fasted state, whereascortisol profiles are measured later in the morning, often in the fedstate. We recommend that patients take their HC on waking, note thetime, have breakfast then provide a blood sample 3-5 hours later formonitoring of HC handling according to the nomogram shown in FIG. 10 b.Additional dosage adjustment, apart from that of weight adjustment, canbe applied accordingly. Optimal HC replacement would be best provided bythe development of a delayed release preparation as suggested for thetreatment of congenital adrenal hyperplasia (Cutler 1996), and attentionshould now be given to the development of such a preparation. TABLE 1Variability Variability in in HC Regime Cmax AUC Bias (AUC) Fixed 10 mg,fasted 31% 50% 6.3% Fixed 10 mg, fed 36% 45% 24.5% BSA-adjusted (5.5mg/m²),  10%*  24%** 7.4% fasted Weight-adjusted (0.12 mg/kg),  7%* 22%** 4.6% fasted¹.¹If weight adjustment was assumed*p < 0.001 vs fixed dose**p < 0.05 vs fixed dose

TABLE 2 p value of each Parameter Covariate covariate r² HC Clearance*Weight <0.001 0.380 Dose 0.042 Oral Volume of Height <0.001 0.421Distribution Dose 0.033 Cmax^(#) BSA <0.001 0.604 Dose <0.001*HC = Hydrocortisone^(#)Cmax = Peak serum cortisol level

TABLE 3 Patient Weight Total dose per 1^(st) dose (kg) day (mg) (mg)2^(nd) dose (mg) 3^(rd) dose (mg) 50 10.0 5.0 2.5 2.5 55-70 15.0 7.5 5.02.5 75-85 17.5 10.0 5.0 2.5 90 20.0 10.0 7.5 2.5  95-110 22.5 12.5 7.52.5 115-120 25.0 15.0 7.5 2.5

Reference List

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1-25. (cancelled)
 26. A method of treating adrenal dysfunction in apatient, said method comprising administering to said patient apharmaceutically effective amount of a combined glucocorticoid and adelivery vehicle, wherein said delivery vehicle provides for delayed andsustained release of said glucorticoid.
 27. A method according to claim26, wherein said glucocorticoid is selected from the group consisting ofhydrocortisone, cortisol, cortisone acetate, prednisolone, prednisone,and dexamethasone.
 28. A method according to claim 27, wherein saidglucocorticoid is hydrocortisone.
 29. A method according to claim 26,wherein the adrenal dysfunction is caused by a condition selected fromthe group consisting of primary and secondary adrenal failure,congenital adrenal hyperplasia, late-onset congenital adrenalhyperplasia, and polycystic ovarian failure.
 30. A method according toclaim 26, wherein the adrenal dysfunction is a congenital adrenaldysfunction.
 31. A method for administering a glucorticoid to an animal,said method comprising the steps of: i) providing a combined preparationof a glucorticoid and a delivery vehicle wherein said vehicle providesfor the sustained release of said glucorticoid; ii) administering thecombined preparation from (i) to an animal requiring treatment such thatthe glucorticoid is released in a sustained manner; iii) providing acombined preparation of a glucorticoid and a delivery vehicle whereinsaid vehicle provides for the delayed and sustained release of theglucorticoid; and iv) administering the combined preparation from (iii)to an animal requiring treatment such that the glucorticoid is releasedin a delayed and sustained manner.
 32. A method according to claim 31,wherein said animal is a human.
 33. A method according to claim 31,wherein said glucocorticoid is selected from the group consisting ofhydrocortisone, cortisol, cortisone acetate, prednisolone, prednisone,and dexamethasone.
 34. A method according to claim 33, wherein saidglucocorticoid is hydrocortisone.
 35. A method according to claim 31,wherein said sustained release preparation is about 10-100 times slowerthan an otherwise identical preparation without the delivery vehicle.36. A method according to claim 35, wherein said sustained releasepreparation is about 30-80 times slower than an otherwise identicalpreparation without the delivery vehicle.
 37. A method according toclaim 36, wherein said sustained release preparation is about 45-50times slower than an otherwise identical preparation without thedelivery vehicle.
 38. A method according to claim 31, wherein thesustained release preparation is administered in the morning, between8:00 am and 12:00 noon.
 39. A method according to claim 31, wherein saiddelayed and sustained release preparation is administered between 8:00pm and 12:00 midnight.
 40. A method of treating congenital adrenalhyperplasia in a patient, said method comprising administering to saidpatient a pharmaceutically effective amount of a combined preparationcomprising cortisol and a delivery vehicle, wherein said deliveryvehicle provides for delayed and sustained release of the cortisol. 41.A method of treating Addison's disease in a patient, said methodcomprising administering to said patient a pharmaceutically effectiveamount of a combined preparation comprising cortisol and a deliveryvehicle, wherein said delivery vehicle provides for delayed andsustained release of the cortisol.